Aromatic-cationic peptides and uses of same

ABSTRACT

The disclosure provides compositions and methods relating to aromatic-cationic peptides. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to subjects in need thereof. For example, the peptides may be administered to subjects in need of a mitochondrial-targeted antioxidant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of and priority to U.S. ProvisionalApplication No. 61/569,120 filed Dec. 9, 2011, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present technology relates generally to compositions and methods ofpreventing or treating disease. In particular, the methods relate to theadministration of aromatic-cationic peptides to a subject in needthereof.

BACKGROUND

The aromatic-cationic peptides disclosed herein are useful intherapeutic applications relating to oxidative damage and cell death.When administered to a mammal in need thereof, the peptides localize tothe mitochondria and improve the integrity and function of theorganelle. Administration of the peptides to a subject in need thereofreduces the number of mitochondria undergoing mitochondrial permeabilitytransition, reduces the level of oxidative damage to cells and tissues,and increases the rate of mitochondrial ATP synthesis.

SUMMARY

In one aspect, the present invention provides an aromatic-cationicpeptide or a pharmaceutically acceptable salt thereof In someembodiments, the salt comprises trifluoroacetate salt or acetate salt.In some embodiments, the peptide is selected from the group consistingof:

6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-D-Phe-NH₂ D-Arg-D-Dmt-D-Lys-L-Phe-NH₂D-Arg-D-Dmt-L-Lys-D-Phe-NH₂ D-Arg-D-Dmt-L-Lys-L-Phe-NH₂D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-Dmt-Lys-NH₂ D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH₂D-Arg-L-Dmt-D-Lys-D-Phe-NH₂ D-Arg-L-Dmt-D-Lys-L-Phe-NH₂D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ D-Arg-Phe-Lys-NH₂ D-Arg-Trp-Lys-NH₂D-Arg-Tyr-Lys-NH₂ Dmt-Arg Dmt-Lys Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Dmt-Lys-Phe Dmt-Lys-Phe Dmt-Lys-Phe-NH₂ Dmt-Phe-Arg-LysH-Arg-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-Lys-NH₂ H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-D-Dmt-NH₂ H-Arg-Dmt-Lys-Phe-NH₂H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L- Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L- Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys- Phe-NH₂H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Cha-Lys-NH₂H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-D-Dmt-Lys-Phe-NH₂H-D-Arg-D-Dmt-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-NH₂H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-Dmt-OHH-D-Arg-Dmt-Phe-Lys-NH₂ H-D-Arg-Dmt-Phe-NH₂H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂ H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂ H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L- phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂ H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂ H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-Lys-Dmt-Phe-NH₂ H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Phe-Dmt-Lys-NH₂H-D-Arg-Phe-Lys-Dmt-NH₂ H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-Dmt-Arg-NH₂H-D-His-L-Dmt-L-Lys-L-Phe-NH₂ H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-Dmt-D-Arg-Lys-Phe-NH₂ H-Dmt-D-Arg-NH₂ H-Dmt-D-Arg-Phe-Lys-NH₂H-Dmt-D-Phe-Arg-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂ H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂ H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂ H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂H-L-His-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂ H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂ H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂ H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂ H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂ H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂H-Lys-D-Arg-Dmt-Phe-NH₂ H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂H-Lys-Dmt-Phe-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-Lys-Phe-D-Arg-Dmt-NH₂H-Lys-Phe-Dmt-D-Arg-NH₂ H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-D-Lys-NH₂ H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂H-Phe-D-Dmt-Arg-Lys-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂ H-Phe-Dmt-Lys-D-Arg-NH₂H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-D-Phe-NH₂L-Arg-D-Dmt-D-Lys-L-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-D-Phe-NH₂L-Arg-D-Dmt-L-Lys-L-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-D-Phe-NH₂L-Arg-L-Dmt-D-Lys-L-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-D-Phe-NH₂L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Dmt-D-Arg-NH₂ Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH₂ Lys-Trp-ArgLys-Trp-D-Arg-NH₂ Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Phe-Lys-Dmt-NH₂Succinic monoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂wherein Cha is cyclohexylalanine.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m=1-3;

(iv)

(v)

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m 1-3;

(iv)

(v)

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the aromatic-cationic peptides have a core structuralmotif of alternating aromatic and cationic amino acids. For example, thepeptide may be a tetrapeptide defined by any of formulas III to VI setforth below:

Aromatic-Cationic-Aromatic-Cationic   (Formula III)

Cationic-Aromatic-Cationic-Aromatic   (Formula IV)

Aromatic-Aromatic-Cationic-Cationic   (Formula V)

Cationic-Cationic-Aromatic-Aromatic   (Formula VI)

wherein, Aromatic is a residue selected from the group consisting of:Phe (F), Tyr (Y), Trp (W), and Cyclohexylalanine (Cha); and Cationic isa residue selected from the group consisting of: Arg (R), Lys (K),Norleucine (Nle), and 2-amino-heptanoic acid (Ahe).

In some embodiments, the peptide is defined by formula VII:

In some embodiments, the peptide is an isomer of formula VII, whereinthe chiral centers of formula III are defined asH—(R)-Arg-(S)-DMT-(S)-Lys-(S)-Phe-NH₂, and wherein stereoisomers aredescribed by the formulas

R—S—S—S

S—R—R—R

S—S—S—S

R—R—R—R

R—R—S—S

S—S—R—R

S—R—S—S

R—S—R—R

R—S—R—S

S—R—S—R

R—R—S—R

S—S—R—S

R—R—R—S

S—S—S—R

R—S—S—R

S—R—R—S

In some embodiments, the peptide is a constitutional isomer of formulaVII selected from the group consisting of:

Arg-Dmt-Lys-Phe-NH₂

Phe-Dmt-Arg-Lys-NH₂

Phe-Lys-Dmt-Arg-NH₂

Dmt-Arg-Lys-Phe-NH₂

Lys-Dmt-Arg-Phe-NH₂

Phe-Dmt-Lys-Arg-NH₂

Arg-Lys-Dmt-Phe-NH₂

Arg-Dmt-Phe-Lys-NH₂

In some embodiments, the peptide is defined by formula VIII:

wherein R is selected from

(i) OMe, and

(ii) H.

In some embodiments, the peptide is defined by formula IX:

wherein R is selected from

(i) F,

(ii) Cl, and

(iii) H.

In some embodiments, the peptide is defined by formula X:

wherein R1-R4 are selected from

(i) Ac, (ii) H, (iii) H, (iv) H,

(i) H, (ii) Ac, (iii) H, (iv) H,

(i) H, (ii) H, (iii) Ac, (iv) H, and

(i) H, (ii) H, (iii) H, (iv) OH.

In one embodiment, the peptide is defined by formula XI:

In some aspects, pharmaceutical compositions are provided herein. Insome embodiments, the pharmaceutical compositions include one or morearomatic-cationic peptides or a pharmaceutically acceptable saltthereof, such as acetate salt or trifluoroacetate salt. In someembodiments, the pharmaceutical composition includes one or morepharmaceutically acceptable carriers.

In one aspect, the disclosure provides a method of reducing the numberof mitochondria undergoing mitochondrial permeability transition (MPT),or preventing mitochondrial permeability transitioning in a mammal inneed thereof, the method comprising administering to the mammal aneffective amount of one or more aromatic-cationic peptides describedherein, or a pharmaceutically salt thereof such as acteate salt ortrifluoroacetate salt. In another aspect, the disclosure provides amethod for increasing the ATP synthesis rate in a mammal in needthereof, the method comprising administering to the mammal an effectiveamount of one or more aromatic-cationic peptides described herein or apharmaceutically salt thereof such as acteate salt or trifluoroacetatesalt. In yet another aspect, the disclosure provides a method forreducing oxidative damage in a mammal in need thereof, the methodcomprising administering to the mammal an effective amount of one ormore aromatic-cationic peptides described herein or a pharmaceuticallysalt thereof such as acteate salt or trifluoroacetate salt.

In some aspects, a method for determining the presence or amount of anaromatic-cationic peptide present in a subject is provided. Typically,the methods include detecting the peptide in a biological sample fromthe subject. In some embodiments, the peptide is detected duringadministration of the peptide to a subject; in some embodiments, thepeptide is detected after administration of the peptide to a subject. Insome embodiments, detecting includes HPLC, for example, reverse phaseHPLC or ion exchange HPLC. In some embodiments, detection includes massspectrometry.

In some embodiments, the biological sample comprises a fluid; in someembodiments, the biological sample comprises a cell. In someembodiments, the biological sample comprises a tissue. In otherembodiments, the biological sample comprises a biopsy.

In some embodiments, the aromatic-cationic peptide that is detected isselected from the group consisting one or more of:

D-Arg-Dmt-Lys-Phe-NH₂ Dmt-D-Arg-Phe-Lys-NH₂ Phe-D-Arg-Dmt-Lys-NH₂6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-D-Phe-NH₂ D-Arg-D-Dmt-D-Lys-L-Phe-NH₂D-Arg-D-Dmt-L-Lys-D-Phe-NH₂ D-Arg-D-Dmt-L-Lys-L-Phe-NH₂D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-Dmt-Lys-NH₂ D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH₂D-Arg-L-Dmt-D-Lys-D-Phe-NH₂ D-Arg-L-Dmt-D-Lys-L-Phe-NH₂D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ D-Arg-Phe-Lys-NH₂ D-Arg-Trp-Lys-NH₂D-Arg-Tyr-Lys-NH₂ Dmt-Arg Dmt-Lys Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Dmt-Lys-Phe Dmt-Lys-Phe Dmt-Lys-Phe-NH₂ Dmt-Phe-Arg-LysH-Arg-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-Lys-NH₂ H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-D-Dmt-NH₂ H-Arg-Dmt-Lys-Phe-NH₂H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L- Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L- Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys- Phe-NH₂H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Cha-Lys-NH₂H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-D-Dmt-Lys-Phe-NH₂H-D-Arg-D-Dmt-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-NH₂H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-Dmt-OHH-D-Arg-Dmt-Phe-Lys-NH₂ H-D-Arg-Dmt-Phe-NH₂H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂ H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂ H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L- phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂ H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂ H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-Lys-Dmt-Phe-NH₂ H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Phe-Dmt-Lys-NH₂H-D-Arg-Phe-Lys-Dmt-NH₂ H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-Dmt-Arg-NH₂H-D-His-L-Dmt-L-Lys-L-Phe-NH₂ H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-Dmt-D-Arg-Lys-Phe-NH₂ H-Dmt-D-Arg-NH₂ H-Dmt-D-Arg-Phe-Lys-NH₂H-Dmt-D-Phe-Arg-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂ H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂ H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂ H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂H-L-His-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂ H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂ H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂ H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂ H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂ H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂H-Lys-D-Arg-Dmt-Phe-NH₂ H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂H-Lys-Dmt-Phe-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-Lys-Phe-D-Arg-Dmt-NH₂H-Lys-Phe-Dmt-D-Arg-NH₂ H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-D-Lys-NH₂ H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂H-Phe-D-Dmt-Arg-Lys-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂ H-Phe-Dmt-Lys-D-Arg-NH₂H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-D-Phe-NH₂L-Arg-D-Dmt-D-Lys-L-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-D-Phe-NH₂L-Arg-D-Dmt-L-Lys-L-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-D-Phe-NH₂L-Arg-L-Dmt-D-Lys-L-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-D-Phe-NH₂L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Dmt-D-Arg-NH₂ Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH₂ Lys-Trp-ArgLys-Trp-D-Arg-NH₂ Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Phe-Lys-Dmt-NH₂Succinic monoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂wherein Cha is cyclohexylalanine.

In some aspects, a kit for the detection of aromatic-cationic peptidesis provided. In some embodiments, the kits include a biological samplecollector to collect a sample from the subject, and a sample storagedevice for preservation of the biological sample. In some embodiments,the biological sample comprises a fluid. In some embodiments, thebiological sample comprises a cell. In some embodiments, the biologicalsample comprises a tissue sample. In some embodiments, the biologicalsample comprises a biopsy.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “biological sample” refers to material derivedfrom or contacted by living cells. The term encompasses tissues, cellsand biological fluids isolated from a subject, as well as tissues, cellsand fluids present within a subject. Biological samples include but arenot limited to, whole blood, fractionated blood, semen, saliva, tears,urine, fecal material, sweat, buccal, skin, cerebrospinal fluid, andhair. Biological samples also includes biopsies of internal organs andcancers. Biological samples can be obtained from subjects for diagnosisor research or can be obtained from undiseased individuals, as controlsor for basic research.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect.In the context of therapeutic or prophylactic applications, the amountof a composition administered to the subject will depend on the type andseverity of the disease and on the characteristics of the individual,such as general health, age, sex, body weight and tolerance to drugs. Itwill also depend on the degree, severity and type of disease. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. The compositions can also be administered incombination with one or more additional therapeutic compounds.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide”, “peptide”, and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. It is also to be appreciatedthat the various modes of treatment or prevention of medical conditionsas described are intended to mean “substantial”, which includes totalbut also less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

Methods of Prevention or Treatment

The present technology relates to the treatment or prevention of diseaseby administration of certain aromatic-cationic peptides.

The aromatic-cationic peptides are water-soluble and highly polar.Despite these properties, the peptides can readily penetrate cellmembranes. The aromatic-cationic peptides typically include a minimum oftwo or three amino acids or a minimum of four amino acids, covalentlyjoined by peptide bonds. The maximum number of amino acids present inthe aromatic-cationic peptides is about twenty amino acids covalentlyjoined by peptide bonds. Suitably, the maximum number of amino acids isabout twelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the a positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. Optimally, the peptide has no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Optimally, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m=1-3;

(iv)

(v)

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m=1-3;

(iv)

(v)

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the aromatic-cationic peptides have a core structuralmotif of alternating aromatic and cationic amino acids. Fr example, thepeptide may be a tetrapeptide defined by any of formulas III to VI setforth below:

Aromatic-Cationic-Aromatic-Cationic   (Formula III)

Cationic-Aromatic-Cationic-Aromatic   (Formula IV)

Aromatic-Aromatic-Cationic-Cationic   (Formula V)

Cationic-Cationic-Aromatic-Aromatic   (Formula VI)

wherein, Aromatic is a residue selected from the group consisting of:Phe (F), Tyr (Y), Trp (W), and Cyclohexylalanine (Cha); and Cationic isa residue selected from the group consisting of: Arg (R), Lys (K),Norleucine (Nle), and 2-amino-heptanoic acid (Ahe).

In one aspect, the disclosure provides a method of reducing the numberof mitochondria undergoing mitochondrial permeability transition (MPT),or preventing mitochondrial permeability transitioning in a mammal inneed thereof, the method comprising administering to the mammal aneffective amount of one or more aromatic-cationic peptides describedherein. In another aspect, the disclosure provides a method forincreasing the ATP synthesis rate in a mammal in need thereof, themethod comprising administering to the mammal an effective amount of oneor more aromatic-cationic peptides described herein. In yet anotheraspect, the disclosure provides a method for reducing oxidative damagein a mammal in need thereof, the method comprising administering to themammal an effective amount of one or more aromatic-cationic peptidesdescribed herein.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal. In one embodiment, thearomatic-cationic peptide may have

(a) at least one net positive charge;

(b) a minimum of three amino acids;

(c) a maximum of about twenty amino acids;

(d) a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and

(e) a relationship between the minimum number of aromatic groups (a) andthe total number of net positive charges (p_(t)) wherein 3a is thelargest number that is less than or equal to p_(t)+1, except that when ais 1, p_(t) may also be 1.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

Aromatic-cationic peptides include, but are not limited to, thefollowing exemplary peptides:

6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-D-Phe-NH₂ D-Arg-D-Dmt-D-Lys-L-Phe-NH₂D-Arg-D-Dmt-L-Lys-D-Phe-NH₂ D-Arg-D-Dmt-L-Lys-L-Phe-NH₂D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-Dmt-Lys-NH₂ D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH₂D-Arg-L-Dmt-D-Lys-D-Phe-NH₂ D-Arg-L-Dmt-D-Lys-L-Phe-NH₂D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ D-Arg-Phe-Lys-NH₂ D-Arg-Trp-Lys-NH₂D-Arg-Tyr-Lys-NH₂ Dmt-Arg Dmt-Lys Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Dmt-Lys-Phe Dmt-Lys-Phe Dmt-Lys-Phe-NH₂ Dmt-Phe-Arg-LysH-Arg-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-Lys-NH₂ H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-D-Dmt-NH₂ H-Arg-Dmt-Lys-Phe-NH₂H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L- Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L- Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys- Phe-NH₂H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Cha-Lys-NH₂H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-D-Dmt-Lys-Phe-NH₂H-D-Arg-D-Dmt-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-NH₂H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-Dmt-OHH-D-Arg-Dmt-Phe-Lys-NH₂ H-D-Arg-Dmt-Phe-NH₂H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂ H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂ H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L- phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂ H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂ H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-Lys-Dmt-Phe-NH₂ H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Phe-Dmt-Lys-NH₂H-D-Arg-Phe-Lys-Dmt-NH₂ H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-Dmt-Arg-NH₂H-D-His-L-Dmt-L-Lys-L-Phe-NH₂ H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-Dmt-D-Arg-Lys-Phe-NH₂ H-Dmt-D-Arg-NH₂ H-Dmt-D-Arg-Phe-Lys-NH₂H-Dmt-D-Phe-Arg-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂ H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂ H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂ H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂H-L-His-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂ H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂ H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂ H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂ H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂ H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂H-Lys-D-Arg-Dmt-Phe-NH₂ H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂H-Lys-Dmt-Phe-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-Lys-Phe-D-Arg-Dmt-NH₂H-Lys-Phe-Dmt-D-Arg-NH₂ H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-D-Lys-NH₂ H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂H-Phe-D-Dmt-Arg-Lys-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂ H-Phe-Dmt-Lys-D-Arg-NH₂H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-D-Phe-NH₂L-Arg-D-Dmt-D-Lys-L-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-D-Phe-NH₂L-Arg-D-Dmt-L-Lys-L-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-D-Phe-NH₂L-Arg-L-Dmt-D-Lys-L-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-D-Phe-NH₂L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Dmt-D-Arg-NH₂ Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH₂ Lys-Trp-ArgLys-Trp-D-Arg-NH₂ Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Phe-Lys-Dmt-NH₂Succinic monoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂wherein Cha is cyclohexylalanine.

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Mu-opioid activity can beassessed by radioligand binding to cloned mu-opioid receptors or bybioassays using the guinea pig ileum (Schiller et al., Eur J Med Chem,35:895-901, 2000; Zhao et al., J Pharmacol Exp Ther, 307:947-954, 2003).Activation of the mu-opioid receptor typically elicits an analgesiceffect. In certain instances, an aromatic-cationic peptide havingmu-opioid receptor agonist activity is preferred. For example, duringshort-term treatment, such as in an acute disease or condition, it maybe beneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. Such acute diseases and conditions are oftenassociated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic-cationic peptide may be beneficial inthe treatment regimen of the human patient or other mammal. Anaromatic-cationic peptide which does not activate the mu-opioidreceptor, however, may also be used with or without an analgesic,according to clinical requirements. Peptides which have mu-opioidreceptor agonist activity are typically those peptides which have atyrosine residue or a tyrosine derivative at the N-terminus (i.e., thefirst amino acid position).

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor agonist activity is preferred. Forexample, during long-term treatment, such as in a chronic disease stateor condition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances, thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatient or other mammal. Potential adverse effects may include sedation,constipation and respiratory depression. In such instances anaromatic-cationic peptide that does not activate the mu-opioid receptormay be an appropriate treatment. Peptides that do not have mu-opioidreceptor agonist activity generally do not have a tyrosine residue or aderivative of tyrosine at the N-terminus (i.e., amino acid position 1).The amino acid at the N-terminus can be any naturally occurring ornon-naturally occurring amino acid other than tyrosine. In oneembodiment, the amino acid at the N-terminus is phenylalanine or itsderivative. Exemplary derivatives of phenylalanine includeT-methylphenylalanine (Mmp), 2′,6′-dimethylphenylalanine (2′,6′-Dmp),N,2′,6′-trimethylphenylalanine (Tmp), and2′-hydroxy-6′-methylphenylalanine (Hmp).

The peptides mentioned herein and their derivatives can further includefunctional variants. A peptide is considered a functional variant if thevariant has the same function as the stated peptide. The analog may, forexample, be a substitution variant of a peptide, wherein one or moreamino acids are substituted by another amino acid. Suitable substitutionvariants of the peptides include conservative amino acid substitutions.Amino acids may be grouped according to their physicochemicalcharacteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

In some embodiments, the peptides disclosed herein are derived fromprecursors, such as peptide precursors. For example, in someembodiments, the precursor comprises an aromatic-cationic which is alsoa therapeutic agent or drug.

Synthesis of Aromatic-Cationic Peptides

The aromatic-cationic peptides disclosed herein may be synthesized byany of the methods well known in the art. Suitable methods forchemically synthesizing the protein include, for example, liquid phaseand solid phase synthesis, and those methods described by Stuart andYoung in Solid Phase Peptide Synthesis, Second Edition, Pierce ChemicalCompany (1984), and in Methods Enzymol., 289, Academic Press, Inc, NewYork (1997). Recombinant peptides may be generated using conventionaltechniques in molecular biology, protein biochemistry, cell biology, andmicrobiology, such as those described in Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N.Y., 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

Detection and Characterization of Aromatic-Cationic Peptides

The aromatic-cationic peptides described herein may be detected and/orcharacterized using methods known in the art. Peptides in a sample maybe detected, for example, using methods of high performance liquidchromatography (HPLC) such as those described in Aguilar, HPLC ofPeptides and Proteins: Methods and Protocols, Humana Press, New Jersey(2004). Peptides may be detected, for example, using reverse-phase HPLC(RP-HPLC) or ion exchange HPLC. High-performance liquid chromatography(or high-pressure liquid chromatography, HPLC) is a chromatographictechnique that can separate a mixture of compounds and is used inbiochemistry and analytical chemistry to identify, quantify and purifythe individual components of the mixture. HPLC typically utilizesdifferent types of stationary phases, a pump that moves the mobilephase(s) and analyte through the column, and a detector to provide acharacteristic retention time for the analyte. The detector may alsoprovide additional information related to the analyte, (e.g., UV/Visspectroscopic data for analyte if so equipped). Analyte retention timevaries depending on the strength of its interactions with the stationaryphase, the ratio/composition of solvent(s) used, and the flow rate ofthe mobile phase. Typically, with HPLC, a pump (rather than gravity)provides the higher pressure required to move the mobile phase andanalyte through a relatively densely packed column. The increaseddensity arises from smaller particle sizes. This allows for a betterseparation on columns of shorter length when compared to ordinary columnchromatography.

In some embodiments, peptides are detected and/or characterized usingreverse phase HPLC (RP-HPLC). Reversed phase HPLC (RP-HPLC or RPC)typically includes a non-polar stationary phase and an aqueous,moderately polar mobile phase. One common stationary phase is a silicawhich has been treated with RMe2SiCl, where R is a straight chain alkylgroup such as C₁₈H₃₇ or C₈H₁₇. With these stationary phases, retentiontime is longer for molecules which are less polar, while polar moleculeselute more readily.

In some emobdiments, peptides are detected and/or characterized usingion exchange HPLC. Typically, in ion-exchange chromatography, retentionis based on the attraction between solute ions and charged sites boundto the stationary phase. Ions of the same charge are excluded. Typicaltypes of ion exchangers include but are not limited to the following.Polystyrene resins. These resins allow cross linkage which increases thestability of the chain. In general, higher cross linkage reducesswerving, which increases the equilibration time and ultimately improvesselectivity. Cellulose and dextran ion exchangers (gels): These possesslarger pore sizes and low charge densities making them suitable forprotein separation. Controlled-pore glass or porous silica.

In general, ion exchangers favor the binding of ions of higher chargeand smaller radius. Typically, an increase in counter ion (with respectto the functional groups in resins) concentration reduces the retentiontime. Typically, an increase in pH reduces the retention time in cationexchange while a decrease in pH reduces the retention time in anionexchange.

In addition, peptides in a sample may be characterized, for example,using methods of mass spectrometry (MS). A general reference related tomethods of mass spectrometry is Sparkman, Mass Spectrometry DeskReference, Pittsburgh: Global View Pub (2000).

One of skill in the art will understand that the aromatic-cationicpeptides described herein may be detected and/or characterized using anynumber of conventional biochemical methods known in the art. The HPLCand MS methods described herein are illustrative and are not to beconstrued as limiting in any way.

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides

The aromatic-cationic peptides described herein are useful to prevent ortreat disease. Specifically, the disclosure provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) disease by administering the aromatic-cationicpeptides described herein. Accordingly, the present methods provide forthe prevention and/or treatment of disease in a subject by administeringan effective amount of an aromatic-cationic peptide to a subject in needthereof.

In one embodiment, the peptides described above are useful in treatingany disease or condition that is associated with mitochondrialpermeability transition (MPT). Reducing the number of mitochondriaundergoing, and preventing, MPT is important, since MPT is associatedwith several common diseases and conditions in mammals. Such diseasesand conditions include, but are not limited to, ischemia and/orreperfusion of a tissue or organ, hypoxia, neurodegenerative diseases,etc. Mammals in need of treatment or prevention of MPT are those mammalssuffering from these diseases or conditions.

Ischemia in a tissue or organ of a mammal is a multifaceted pathologicalcondition which is caused by oxygen deprivation (hypoxia) and/or glucose(e.g., substrate) deprivation. Oxygen and/or glucose deprivation incells of a tissue or organ leads to a reduction or total loss of energygenerating capacity and consequent loss of function of active iontransport across the cell membranes. Oxygen and/or glucose deprivationalso leads to pathological changes in other cell membranes, includingpermeability transition in the mitochondrial membranes. In additionother molecules, such as apoptotic proteins normally compartmentalizedwithin the mitochondria, may leak out into the cytoplasm and causeapoptotic cell death. Profound ischemia can lead to necrotic cell death.Ischemia or hypoxia in a particular tissue or organ may be caused by aloss or severe reduction in blood supply to the tissue or organ. Theloss or severe reduction in blood supply may, for example, be due tothromboembolic stroke, coronary atherosclerosis, or peripheral vasculardisease. The tissue affected by ischemia or hypoxia is typically muscle,such as cardiac, skeletal, or smooth muscle. The organ affected byischemia or hypoxia may be any organ that is subject to ischemia orhypoxia. Examples of organs affected by ischemia or hypoxia includebrain, heart, kidney, and prostate. For instance, cardiac muscleischemia or hypoxia is commonly caused by atherosclerotic or thromboticblockages which lead to the reduction or loss of oxygen delivery to thecardiac tissues by the cardiac arterial and capillary blood supply. Suchcardiac ischemia or hypoxia may cause pain and necrosis of the affectedcardiac muscle, and ultimately may lead to cardiac failure. Ischemia orhypoxia in skeletal muscle or smooth muscle may arise from similarcauses. For example, ischemia or hypoxia in intestinal smooth muscle orskeletal muscle of the limbs may also be caused by atherosclerotic orthrombotic blockages.

Reperfusion is the restoration of blood flow to any organ or tissue inwhich the flow of blood is decreased or blocked. For example, blood flowcan be restored to any organ or tissue affected by ischemia or hypoxia.The restoration of blood flow (reperfusion) can occur by any methodknown to those in the art. For instance, reperfusion of ischemic cardiactissues may arise from angioplasty, coronary artery bypass graft, or theuse of thrombolytic drugs.

The methods described herein can also be used in the treatment orprophylaxis of neurodegenerative diseases associated with MPT.Neurodegenerative diseases associated with MPT include, for instance,Parkinson's disease, Alzheimer's disease, Huntington's disease andAmyotrophic Lateral Sclerosis (ALS, also known as Lou Gherig's disease).The methods disclosed herein can be used to delay the onset or slow theprogression of these and other neurodegenerative diseases associatedwith MPT. The methods disclosed herein are particularly useful in thetreatment of humans suffering from the early stages of neurodegenerativediseases associated with MPT and in humans predisposed to thesediseases.

The aromatic-cationic peptides described above are also useful inpreventing or treating insulin resistance, metabolic syndrome, burninjuries and secondary complications, heart failure, diabeticcomplications (such as diabetic retinopathy), ophthalmic conditions(such as choroidal neovascularization, retinal degeneration, andoxygen-induced retinopathy).

The aromatic-cationic peptides described above are also useful inreducing oxidative damage in a mammal in need thereof. Mammals in needof reducing oxidative damage are those mammals suffering from a disease,condition or treatment associated with oxidative damage. Typically, theoxidative damage is caused by free radicals, such as reactive oxygenspecies (ROS) and/or reactive nitrogen species (RNS). Examples of ROSand RNS include hydroxyl radical (HO.), superoxide anion radical (O₂ ⁻),nitric oxide (NO.), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl⁻)and peroxynitrite anion (ONOO⁻). In one embodiment, a mammal in needthereof may be a mammal undergoing a treatment associated with oxidativedamage. For example, the mammal may be undergoing reperfusion, ischemia,or hypoxia.

In another embodiment, the aromatic-cationic peptides can be used toprevent lipid peroxidation and/or inflammatory processes that areassociated with oxidative damage for a disease or condition. Lipidperoxidation refers to oxidative modification of lipids. The lipids canbe present in the membrane of a cell. This modification of membranelipids typically results in change and/or damage to the membranefunction of a cell. In addition, lipid peroxidation can also occur inlipids or lipoproteins exogenous of a cell. For example, low-densitylipoproteins are susceptible to lipid peroxidation. An example of acondition associated with lipid peroxidation is atherosclerosis.Reducing oxidative damage associated with atherosclerosis is importantsince atherosclerosis is implicated in, for example, heart attacks andcoronary artery disease.

Inflammatory processes include and activation of the immune system.Typically, the immune system is activated by an antigenic substance. Theantigenic substance can be any substance recognized by the immunesystem, and include self-derived particles and foreign-derivedparticles. Examples of diseases or conditions occurring from aninflammatory process to self-derived particles include arthritis andmultiple sclerosis. Examples of foreign particles include viruses andbacteria. The virus can be any virus which activates an inflammatoryprocess, and associated with oxidative damage. Examples of virusesinclude, hepatitis A, B or C virus, human immunodeficiency virus,influenza virus, and bovine diarrhea virus. For example, hepatitis viruscan elicit an inflammatory process and formation of free radicals,thereby damaging the liver. The bacteria can be any bacteria, andinclude gram-negative or gram-positive bacteria. Gram-negative bacteriacontain lipopolysaccharide in the bacteria wall. Examples ofgram-negative bacteria include Escherichia coli, Klebsiella pneumoniae,Proteus species, Pseudomonas aeruginosa, Serratia, and Bacteroides.Examples of gram-positive bacteria include pneumococci and streptococci.An example of an inflammatory process associated with oxidative stresscaused by a bacteria is sepsis. Typically, sepsis occurs whengram-negative bacteria enter the bloodstream.

Liver damage caused by a toxic agent is another condition associatedwith an inflammatory process and oxidative stress. The toxic agent canbe any agent which causes damage to the liver. For example, the toxicagent can cause apoptosis and/or necrosis of liver cells. Examples ofsuch agents include alcohol, and medication, such as prescription andnon-prescription drugs taken to treat a disease or condition.

The methods disclosed herein can also be used in reducing oxidativedamage associated with any neurodegenerative disease or condition. Theneurodegenerative disease can affect any cell, tissue or organ of thecentral and peripheral nervous system. Examples of such cells, tissuesand organs include, the brain, spinal cord, neurons, ganglia, Schwanncells, astrocytes, oligodendrocytes and microglia. The neurodegenerativecondition can be an acute condition, such as a stroke or a traumaticbrain or spinal cord injury. In another embodiment, theneurodegenerative disease or condition can be a chronicneurodegenerative condition. In a chronic neurodegenerative condition,the free radicals can, for example, cause damage to a protein. Anexample of such a protein is amyloid .beta.-protein. Examples of chronicneurodegenerative diseases associated with damage by free radicalsinclude Parkinson's disease, Alzheimer's disease, Huntington's diseaseand Amyotrophic Lateral Sclerosis (also known as Lou Gherig's disease).

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic. In various embodiments, suitable in vitro orin vivo assays are performed to determine the effect of a specificaromatic-cationic peptide-based therapeutic and whether itsadministration is indicated for treatment. In various embodiments, invitro assays can be performed with representative animal models, todetermine if a given aromatic-cationic peptide-based therapeutic exertsthe desired effect in preventing or treating a disease or medicalcondition. Compounds for use in therapy can be tested in suitable animalmodel systems including, but not limited to rats, mice, chicken, pigs,cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal modelsystems known in the art can be used prior to administration to humansubjects.

Prophylactic Methods. In one aspect, the invention provides a method forpreventing, in a subject, disease by administering to the subject anaromatic-cationic peptide that prevents the initiation or progression ofthe condition. In prophylactic applications, pharmaceutical compositionsor medicaments of aromatic-cationic peptides are administered to asubject susceptible to, or otherwise at risk of a disease or conditionin an amount sufficient to eliminate or reduce the risk, lessen theseverity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease. Administration of a prophylactic aromatic-cationic canoccur prior to the manifestation of symptoms characteristic of theaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. The appropriate compound canbe determined based on screening assays described above.

Therapeutic Methods. Another aspect of the technology includes methodsof treating disease in a subject for therapeutic purposes. Intherapeutic applications, compositions or medicaments are administeredto a subject suspected of, or already suffering from such a disease inan amount sufficient to cure, or at least partially arrest, the symptomsof the disease, including its complications and intermediatepathological phenotypes in development of the disease. As such, theinvention provides methods of treating an individual afflicted with adisease or medical condition.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, suitably a human. When used in vivo fortherapy, the aromatic-cationic peptides are administered to the subjectin effective amounts (i.e., amounts that have desired therapeuticeffect). The dose and dosage regimen will depend upon the degree of theinjury in the subject, the characteristics of the particulararomatic-cationic peptide used, e.g., its therapeutic index, thesubject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods may be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide may be administeredsystemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the salt comprises trifluoroacetatesalt or acetate salt.

The aromatic-cationic peptides described herein or a pharmaceuticallysalt thereof such as acteate salt or trifluoroacetate salt, can beincorporated into pharmaceutical compositions for administration, singlyor in combination, to a subject for the treatment or prevention of adisease or medical condition described herein. Such compositionstypically include the active agent and a pharmaceutically acceptablecarrier. As used herein the term “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions or a pharmaceutically saltthereof such as acteate salt or trifluoroacetate salt, can include acarrier, which can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like.Glutathione and other antioxidants can be included to prevent oxidation.In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate orgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic peptide or a pharmaceutically salt thereof such as acteatesalt or trifluoroacetate salt, can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Dosage may also be determined empirically by detecting aromatic-cationicpeptides in a biological sample from a subject. Biological samplesobtained from a subject who has been administered aromatic-cationicpeptides may be subjected to HPLC and/or MS to detect and characterizethe aromatic-cationic peptides present in the subject's bodily fluidsand tissues. Biological samples include any material derived from orcontacted by living cells. Examples of biological samples include butare not limited to whole blood, fractionated blood, semen, saliva,tears, urine, fecal material, sweat, buccal, skin, cerebrospinal fluid,and hair. Biological samples also include biopsies of internal organs,organs removed for transplant or cancers. The presence ofaromatic-cationic peptides in the biological sample is established bycomparison to data obtained for reference samples such as those providedin Example 6. The levels of aromatic-cationic peptides present in thesample may serve as a basis to increase or decrease the dosage of anaromatic-cationic peptide or a precursor thereof, administered to agiven subject, wherein the precursor may be an aromatic-cationic whichis also a therapeutic agent or drug.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Preferably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.1-10,000 micrograms per kg body weight. In one embodiment,aromatic-cationic peptide concentrations in a carrier range from 0.2 to2000 micrograms per delivered milliliter. An exemplary treatment regimeentails administration once per day or once a week. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.01 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.01 to about 0.5 mg/kg/h, suitably fromabout 0.01 to about 0.1 mg/kg/h. In one embodiment, the mid-dose isprovided from about 0.1 to about 1.0 mg/kg/h, suitably from about 0.1 toabout 0.5 mg/kg/h. In one embodiment, the high dose is provided fromabout 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2mg/kg/h.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

In some embodiments, multiple doses, or varying amounts of anaromatic-cationic peptide are administered to a subject. In someembodiments, the multiple doses or varying amounts of the peptide areadministered throughout the course of a procedure (e.g., a surgery) orare administered throughout the course of a disease or conditions. Forexample, in some embodiments, the peptide is administeredintraveneously, for example during a surgery, and the amount of peptideprovided to the subject is adjusted during the procedure. In otherembodiments, the subject is administered a dose of peptide (e.g., orallyor by injection, e.g., intradermal, subcutaneous, intramuscular,intravenous, intraosseous, and intraperitoneal) about once every 10minutes, 15 minutes, 30 minutes, hour, 2 hours, 4 hours, 6 hours, 8hours, 10 hours, 12 hours, once per day, once every other day, or onceper week. In some embodiments, the amount of peptide present in thesubject (the subject's peptide level) is monitored to determine theappropriate dose and schedule needed to maintain a desired peptide levelin the subject. In some embodiments, peptide levels are determinedperiodically during administration, and/or are determined at one or moretime points after administration. For example, in some embodiments,peptide levels are determined within a few minutes of administration,about 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6hours, 8 hours, 10 hours, 12 hours, 24 hours, two days, 3 days 5 days, 7days or 14 days after administration. In other embodiments, the subjectspeptide levels are determined every 10 minutes, 15 minutes, 20 minutes,30 minutes, hourly, every two hours, every 4 hours, every 6 hours orevery 12 hours, for a predetermined time, such as during a surgicalprocedure. Depending on the determined peptide level, one or moreadditional doses may be provided to achieve a desired peptide level tomaintain a therapeutic effect. In some embodiments, peptide levels maybe found sufficient to delay an additional dose or doses.

In some embodiments, the detected levels of aromatic-cationic peptideare compared to aromatic-cationic peptide levels in a healthy controlsubject (e.g., a subject who has been administered substantially thesame dose of the peptide via substantially the same route ofadministration). Additionally or alternatively, in some embodiments, thelevel of the peptide is determined in different organs, systems and/orfluids of a subject. In some embodiments, the level of the peptide isdetermined in different organs, systems and/or fluids of a subject andcompared to peptide levels in comparable systems, organs, and fluids ofa control subject. In some embodiments, such an analysis providesinformation regarding the availability of the peptide, and the transportof the peptide throughout the body of the subject as compared to thecontrol. For example, the route of administration may be changed for aparticular subject to optimize peptide delivery to a particular tissueor organ (e.g., to achieve a more localized distribution of thepeptide). Additionally or alternatively, the route of administrationcould be changed for a particular subject to for a more systemic peptidedistribution.

Method for identifying and determining the presence or amount (level) ofan aromatic-cationic peptide in a subject sample are known in the artand include, but are not limited to HPLC and mass spectrometry.

As noted previously, in some embodiments, peptide levels are determinedin a biological sample from the subject, and include, withoutlimitation, blood samples, tissues samples, (e.g., organ or tumor biopsysamples), urine and saliva.

Also disclosed herein are kits for the detection of aromatic-cationicpeptides. In some embodiments, the kits include a sample collectiondevice to collect a sample from the subject, and a sample storage devicefor preservation of the biological sample. Depending on the intendedmethod of detection, the sample is stored in an appropriate buffer orpreservative, also provided in the kit. In some embodiments, a samplecollector includes a container for liquid, such as a vial or tube (e.g.,for blood, blood products, urine). In other embodiments, the samplecollector is an absorbent material, such as a sterile cotton swab (e.g.,to collect a buccal sample, saliva, nasal swabs, etc), a slide, asterile paper, a card, a syringe, etc. The sample storage device may beany device that will encase and protect the sample during transport,shipment and/or storage. For example, in some embodiments, the samplestorage device is a sealable tube.

In some embodiments, the kits also include instructions for obtaining asample and properly storing the sample until analysis. In someembodiments, the sample includes a bodily fluid, one or more cells, atissue or portion of an organ, a biopsy sample, and/and a portion of atumor.

The mammal treated in accordance with the present methods can be anymammal, including, for example, farm animals, such as sheep, pigs, cows,and horses; pet animals, such as dogs and cats; laboratory animals, suchas rats, mice and rabbits. In a suitable embodiment, the mammal is ahuman.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Aromatic-Cationic Peptides of the Present Technology InhibitInhibits H₂O₂ Generation by Isolated Mitochondria

In this Example, the effects of the aromatic-cationic peptides of theinvention on H₂O₂ generation by isolated mitochondria are investigated.H₂O₂ is measured using luminol chemiluminescence as described in Y. Li,H. Zhu, M. A. Trush, Biochim. Biophys. Acta 1428, 1-12 (1999)). Briefly,0.1 mg mitochondrial protein is added to 0.5 ml potassium phosphatebuffer (100 mM, pH 8.0) in the absence or presence of anaromatic-cationic peptide. Luminal (25 mM) and 0.7 IU horseradishperoxidase are added, and chemilumunescence is monitored with aChronolog Model 560 aggregometer (Havertown, Pa.) for 20 min at 37° C.The amount of H₂O₂ produced is quantified as the area under the curve(AUC) over 20 min, and all data are normalized to AUC produced bymitochondria alone.

It is predicted that the aromatic-cationic peptide will reduce thespontaneous production of H₂O₂ by isolated mitochondria. As such, thearomatic-cationic peptides are useful for reducing oxidative damage andare useful in the treatment or prevention of diseases or conditions thatrelate to oxidative damage.

Example 2 Aromatic-Cationic Peptides of the Present Technology ReduceIntracellular ROS and Increase Cell Survival

To show that the claimed peptides are effective antioxidants whenapplied to whole cells, neuronal N₂A cells are plated in 96-well platesat a density of 1×10⁴/well and allowed to grow for 2 days beforetreatment with tBHP (0.5 or 1 mM) for 40 mM. Cells are washed twice andreplaced with medium alone or medium containing varying concentrationsof aromatic-cationic peptides (10⁻¹² M to 10⁻⁹ M) for 4 h. IntracellularROS is measured by carboxy-H₂DCFDA (Molecular Probes, Portland, Oreg.).Cell death is assessed by a cell proliferation assay (MTS assay,Promega, Madison, Wis.).

Incubation with tBHP will result in an increase in intracellular ROS anddecrease in cell viability. However, it is predicted that incubation ofthese cells with an aromatic-cationic peptide will reduce intracellularROS and increase cell survival. As such, the aromatic-cationic peptidesare useful for reducing oxidative damage and are useful in the treatmentor prevention of diseases or conditions that relate to oxidative damage.

Example 3 Aromatic-Cationic Peptides of the Present Technology ProtectAgainst MPT Induced by Ca²⁺ and 3-Nitropropionic Acid

To isolate mitochondria from mouse liver, mice are sacrificed bydecapitation. The liver is removed and rapidly placed into chilled liverhomogenization medium. The liver is finely minced using scissors andthen homogenized by hand using a glass homogenizer. The homogenate iscentrifuged for 10 min at 1000 g at 4° C. The supernatant is aspiratedand transferred to polycarbonate tubes and centrifuged again for 10 mMat 3000 g, 4° C. The resulting supernatant is removed, and the fattylipids on the side-wall of the tube are carefully wiped off. The pelletis resuspended in liver homogenate medium and the homogenizationrepeated twice. The final purified mitochondrial pellet is resuspendedin medium. Protein concentration in the mitochondrial preparation isdetermined by the Bradford procedure.

To investigate the localization of the aromatic-cationic peptides of theinvention, approximately 1.5 mg mitochondria in 400 μl buffer isincubated with labeled aromatic-cationic peptide for 5-30 min at 37° C.The mitochondria are then centrifuged down and the amount of label ismeasured in the mitochondrial fraction and buffer fraction. Assuming amitochondrial matrix volume of 0.7 ul/mg protein (Lim et al., J Physiol545:961-974, 2002), the concentration of peptide in mitochondria can bedetermined. It is predicted that the claimed aromatic-cationic peptideswill be more concentrated in mitochondria compared to the bufferfraction.

To investigate the effects of the aromatic-cationic peptides of theinvention on mitochondrial membrane potential, isolated mouse livermitochondria are incubated with 100-200 μM aromatic-cationic peptide.Mitochondrial membrane potential is measured using tetramethyl rhodaminemethyl ester (TMRM). Addition of mitochondria results in immediatequenching of the TMRM signal which is readily reversed by the additionof FCCP, indicating mitochondrial depolarization. The addition of Ca²⁺(150 μM) results in immediate depolarization followed by progressiveloss of quenching, indicative of MPT. Addition of aromatic-cationicpeptide alone, even at 200 μM, is not predicted to cause mitochondrialdepolarization or MPT. It is also predicted that the aromatic-cationicpeptides will not alter mitochondrial function, including oxygenconsumption during state 3 or state 4, or the respiratory ratio (state3/state 4).

To show that the claimed peptides are effective at protecting againstMPT induced by Ca²⁺ overload, isolated mitochondria are pre-treated witharomatic-cationic peptide (10 μM) for 2 min prior to addition of Ca²⁺.It is predicted that the aromatic-cationic peptides of the inventionwill increase the tolerance of mitochondria to cumulative Ca²⁺challenges.

3-Nitropropionic acid (3NP) is an irreversible inhibitor of succinatedehydrogenase in complex II of the electron transport chain. Addition of3NP (1 mM) to isolated mitochondria causes dissipation of mitochondrialpotential and onset of MPT. Pretreatment of mitochondria with thearomatic-cationic peptides of the invention is predicted to delay theonset of MPT induced by 3NP.

To demonstrate that the aromatic-cationic peptides of the invention canpenetrate cell membranes and protect against mitochondrialdepolarization elicited by 3NP, Caco-2 cells are treated with 3NP (10mM) in the absence or presence of the aromatic-cationic peptides for 4h, and then incubated with TMRM and examined under LSCM. In controlcells, the mitochondria are clearly visualized as fine streaksthroughout the cytoplasm. In cells treated with 3NP, the TMRMfluorescence is much reduced, indicating generalized depolarization. Incontrast, it is predicted that concurrent treatment with thearomatic-cationic peptides of the invention will protect againstmitochondrial depolarization caused by 3NP.

As such, the aromatic-cationic peptides are useful for preventing MPTand are useful in the treatment or prevention of diseases or conditionsthat relate to MPT.

Example 4 Aromatic-Cationic Peptides of the Present Technology ProtectAgainst Mitochondrial Swelling and Cytochrome c Release

MPT pore opening results in mitochondrial swelling. This Exampleexamines the effects of the aromatic-cationic peptides of the inventionon mitochondrial swelling by measuring reduction in absorbance at 540 nm(A₅₄₀). Once the absorbance is measured, the mitochondrial suspension isthen centrifuged and cytochrome c in the mitochondrial pellet andsupernatant is determined by a commercially-available ELISA kit. It ispredicted that pretreatment of isolated mitochondria with thearomatic-cationic peptides of the invention will inhibit swelling andcytochrome c release induced by Ca²⁺ overload. Besides preventing MPTinduced by Ca²⁺ overload, it is predicted that the aromatic-cationicpeptides of the invention will also prevent mitochondrial swellinginduced by MPP⁺ (1-methyl-4-phenylpyridium ion), an inhibitor of complexI of the mitochondrial electron transport chain.

As such, the aromatic-cationic peptides are useful for preventing MPTand are useful in the treatment or prevention of diseases or conditionsthat relate to MPT.

Example 5 The Peptides of the Present Technology Increase the Rate ATPSynthesis in Isolated Mitochondria

This example will demonstrate the impact of peptides of the presenttechnology on the rate of mitochondrial ATP synthesis.

The rate of mitochondrial ATP synthesis will be determined by measuringATP in respiration buffer collected from isolated mitochondria 1 minafter addition of 400 mM ADP. ATP will be assayed by HPLC. Allexperiments will be carried out in triplicate, with n=3. It is predictedthat addition of peptides of the present technology to isolatedmitochondria will increase the rate of ATP synthesis in a dose-dependentmanner.

This result will demonstrate the peptides of the present technology areuseful in methods and compositions for increasing the rate ofmitochondrial ATP synthesis.

Example 6 Characterization of Aromatic-Cationic Peptides

Aromatic-cationic peptides of the present technology can be synthesizedusing solid phase synthesis and characterized using HPLC and MS.Exemplary HPLC and MS methods are provided in Examples 7 and 8 below.

Example 7 Detection of Aromatic-Cationic Peptides in a Biological Sample

This example demonstrates the detection of aromatic-cationic peptides ina biological sample by HPLC. Biological samples are collected fromsubjects in a suitable manner depending on the nature of the sample.Biological samples include any material derived from or contacted byliving cells. Examples of biological samples include but are not limitedto whole blood, fractionated blood, semen, saliva, tears, urine, fecalmaterial, sweat, buccal, skin, cerebrospinal fluid, and hair. Biologicalsamples also include biopsies of internal organs or cancers. Onceobtained, the biological samples are stored in a manner compatible withthe methods of detection until the methods are performed to ensure thepreservation of aromatic-cationic peptides present in the sample.

Samples are loaded onto a 250×4.6 (i.d.) mm C18 5 μm column andsubjected to a gradient of 0.1% trifluoroacetic acid in acetonitrile(Solution A) and 0.1% trifluoroacetic acid in HPLC-grade water (SolutionB) according to the following scheme:

TABLE 6 HLPC Methods A B 0.01 min  7% 93%   25 min 32% 68% 25.1 min100%   0% Flow rate 1.0 ml/min Wave Length 220 nm Load Volume 10 μl

The presence of aromatic-cationic peptides in the biological sample isestablished by comparison to data obtained for reference samples such asthose provided in Example 6.

The foregoing method is illustrative only, and should not be construedas limiting in any way. One of skill in the art will understand that thearomatic-cationic peptides described herein may be analyzed by a numberof HPLC methods, such as those describe in Aguilar, HPLC of Peptides andProteins: Methods and Protocols, Humana Press, New Jersey (2004).

Example 8 Detection of Aromatic-Cationic Peptides in a Biological Sampleby MS

This example demonstrates the detection of aromatic-cationic peptides ina biological sample by MS. Biological samples are collected fromsubjects in a suitable manner depending on the nature of the sample.Biological samples include any material derived from or contacted byliving cells. Examples of biological samples include but are not limitedto whole blood, fractionated blood, semen, saliva, tears, urine, fecalmaterial, sweat, buccal, skin, cerebrospinal fluid, and hair. Biologicalsamples also include biopsies of internal organs or cancers. Onceobtained, the biological samples are stored in a manner compatible withthe methods of detection until the methods are performed to ensure thepreservation of aromatic-cationic peptides present in the sample.

Samples are loaded in a 20 μl volume and analyzed under the followingexemplary conditions.

TABLE 7 MS Methods Probe ESI Nebulizer Gas Flow 1.5 L/min CurvedDesolvation −20.0 v Line (CDL) CDL Temp 250° C. Block Temp 200° C. ProbeBias +4.5 kv Detector 1.5 kv T. Flow 0.2 ml/min Buffer 50% H₂O-50%Acetonitrile

One of skill in the art will understand, that the aromatic-cationicpeptides described herein may be analyzed by a number of MS methods,such as those describe in Sparkman, Mass Spectrometry Desk Reference,Pittsburgh: Global View Pub (2000).

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

1. An aromatic-cationic peptide selected from the group consisting of:6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-D-Phe-NH₂ D-Arg-D-Dmt-D-Lys-L-Phe-NH₂D-Arg-D-Dmt-L-Lys-D-Phe-NH₂ D-Arg-D-Dmt-L-Lys-L-Phe-NH₂D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-Dmt-Lys-NH₂ D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH₂D-Arg-L-Dmt-D-Lys-D-Phe-NH₂ D-Arg-L-Dmt-D-Lys-L-Phe-NH₂D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ D-Arg-Phe-Lys-NH₂ D-Arg-Trp-Lys-NH₂D-Arg-Tyr-Lys-NH₂ Dmt-Arg Dmt-Lys Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Dmt-Lys-Phe Dmt-Lys-Phe Dmt-Lys-Phe-NH₂ Dmt-Phe-Arg-LysH-Arg-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-Lys-NH₂ H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-D-Dmt-NH₂ H-Arg-Dmt-Lys-Phe-NH₂H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH₂H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Cha-Lys-NH₂H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-D-Dmt-Lys-Phe-NH₂H-D-Arg-D-Dmt-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-NH₂H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-Dmt-OHH-D-Arg-Dmt-Phe-Lys-NH₂ H-D-Arg-Dmt-Phe-NH₂H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂ H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂ H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-Lys-Dmt-Phe-NH₂ H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Phe-Dmt-Lys-NH₂H-D-Arg-Phe-Lys-Dmt-NH₂ H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-Dmt-Arg-NH₂H-D-His-L-Dmt-L-Lys-L-Phe-NH₂ H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-Dmt-D-Arg-Lys-Phe-NH₂ H-Dmt-D-Arg-NH₂ H-Dmt-D-Arg-Phe-Lys-NH₂H-Dmt-D-Phe-Arg-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂ H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂ H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂ H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂H-L-His-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂ H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂ H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂ H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂ H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂ H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂H-Lys-D-Arg-Dmt-Phe-NH₂ H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂H-Lys-Dmt-Phe-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-Lys-Phe-D-Arg-Dmt-NH₂H-Lys-Phe-Dmt-D-Arg-NH₂ H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-D-Lys-NH₂ H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂H-Phe-D-Dmt-Arg-Lys-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂ H-Phe-Dmt-Lys-D-Arg-NH₂H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-D-Phe-NH₂L-Arg-D-Dmt-D-Lys-L-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-D-Phe-NH₂L-Arg-D-Dmt-L-Lys-L-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-D-Phe-NH₂L-Arg-L-Dmt-D-Lys-L-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-D-Phe-NH₂L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Dmt-D-Arg-NH₂ Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH₂ Lys-Trp-ArgLys-Trp-D-Arg-NH₂ Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Phe-Lys-Dmt-NH₂Succinic monoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂


2. A pharmaceutical composition comprising one or more aromatic-cationicpeptides of claim 1 and pharmaceutically acceptable salts thereof. 3.The pharmaceutical composition of claim 2 further comprising apharmaceutically acceptable carrier.
 4. A method of reducing the numberof mitochondria undergoing mitochondrial permeability transition (MPT),or preventing mitochondrial permeability transitioning in a mammal inneed thereof, the method comprising administering to the mammal aneffective amount of one or more aromatic-cationic peptides of claim 1.5. A method for reducing oxidative damage in a mammal in need thereof,the method comprising administering to the mammal an effective amount ofone or more aromatic-cationic peptides of claim
 1. 6. A method forincreasing the ATP synthesis rate in a mammal in need thereof, themethod comprising administering to the mammal an effective amount of oneor more aromatic-cationic peptides of claim
 1. 7. A method fordetermining the presence or amount of an administered aromatic-cationicpeptide in a subject, the method comprising: detecting the administeredaromatic-cationic peptide in a biological sample from the subject,wherein the aromatic-cationic peptide is selected from the groupconsisting of: 6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-D-Phe-NH₂ D-Arg-D-Dmt-D-Lys-L-Phe-NH₂D-Arg-D-Dmt-L-Lys-D-Phe-NH₂ D-Arg-D-Dmt-L-Lys-L-Phe-NH₂D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-Dmt-Lys-NH₂ D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH₂D-Arg-L-Dmt-D-Lys-D-Phe-NH₂ D-Arg-L-Dmt-D-Lys-L-Phe-NH₂D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ D-Arg-Phe-Lys-NH₂ D-Arg-Trp-Lys-NH₂D-Arg-Tyr-Lys-NH₂ Dmt-Arg Dmt-Lys Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Dmt-Lys-Phe Dmt-Lys-Phe Dmt-Lys-Phe-NH₂ Dmt-Phe-Arg-LysH-Arg-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-Lys-NH₂ H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-D-Dmt-NH₂ H-Arg-Dmt-Lys-Phe-NH₂H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH₂H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Cha-Lys-NH₂H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-D-Dmt-Lys-Phe-NH₂H-D-Arg-D-Dmt-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-NH₂H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-Dmt-OHH-D-Arg-Dmt-Phe-Lys-NH₂ H-D-Arg-Dmt-Phe-NH₂H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂ H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂ H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂ H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-Lys-Dmt-Phe-NH₂ H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Phe-Dmt-Lys-NH₂H-D-Arg-Phe-Lys-Dmt-NH₂ H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-Dmt-Arg-NH₂H-D-His-L-Dmt-L-Lys-L-Phe-NH₂ H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-Dmt-D-Arg-Lys-Phe-NH₂ H-Dmt-D-Arg-NH₂ H-Dmt-D-Arg-Phe-Lys-NH₂H-Dmt-D-Phe-Arg-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂ H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂ H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂ H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂H-L-His-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂ H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂ H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂ H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂ H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂ H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂H-Lys-D-Arg-Dmt-Phe-NH₂ H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂H-Lys-Dmt-Phe-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-Lys-Phe-D-Arg-Dmt-NH₂H-Lys-Phe-Dmt-D-Arg-NH₂ H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-D-Lys-NH₂ H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂H-Phe-D-Dmt-Arg-Lys-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂ H-Phe-Dmt-Lys-D-Arg-NH₂H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-D-Phe-NH₂L-Arg-D-Dmt-D-Lys-L-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-D-Phe-NH₂L-Arg-D-Dmt-L-Lys-L-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-D-Phe-NH₂L-Arg-L-Dmt-D-Lys-L-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-D-Phe-NH₂L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Dmt-D-Arg-NH₂ Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH₂ Lys-Trp-ArgLys-Trp-D-Arg-NH₂ Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Phe-Lys-Dmt-NH₂Succinic monoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂


8. The method of claim 7, wherein detecting is performed duringadministration of the peptide.
 9. The method of claim 7, whereindetecting is performed after administration of the peptide.
 10. Themethod of any one of claim 7, wherein detecting comprises HPLC.
 11. Themethod of claim 10, wherein the HPLC comprises reverse phase HPLC. 12.The method of claim 10, wherein the HPLC comprises ion exchange HPLC.13. The method of claim 7, wherein detecting comprises massspectrometry.
 14. The method of claim 7, wherein the biological samplecomprises a fluid.
 15. The method of claim 7, wherein the biologicalsample comprises a cell.
 16. The method of claim 7, wherein thebiological sample comprises a tissue.
 17. The method of any one ofclaims 7, wherein the biological sample comprises a biopsy.
 18. Anaromatic-cationic peptide selected from the group consisting of: a) anaromatic-cationic peptide comprising formula VII or a stereoisomerthereof

wherein the chiral centers of formula III are defined asH—(R)-Arg-(S)-DMT-(S)-Lys-(S)-Phe-NH₂, and wherein stereoisomers aredescribed by the formulas R—S—S—S, S—R—R—R, S—S—S—S, R—R—R—R, R—R—S—S,S—S—R—R, S—R—S—S, R—S—R—R, R—S—R—S, S—R—S—R, R—R—S—R, S—S—R—S, R—R—R—S,S—S—S—R, R—S—S—R, and S—R—R—S; b) an aromatic-cationic peptidecomprising formula VII or a constitutional thereof

selected from the group consisting of Arg-Dmt-Lys-Phe-NH₂,Phe-Dmt-Arg-Lys-NH₂, Phe-Lys-Dmt-Arg-NH₂, Dmt-Arg-Lys-Phe-NH₂,Lys-Dmt-Arg-Phe-NH₂, Phe-Dmt-Lys-Arg-NH₂, Arg-Lys-Dmt-Phe-NH₂, orArg-Dmt-Phe-Lys-NH₂; c) an aromatic-cationic peptide comprising formulaVIII

wherein R is selected from (i) OMe, and (ii) H, d) an aromatic-cationicpeptide comprising formula IX

wherein R is selected from (i) F, (ii) Cl, and (iii) H, e) anaromatic-cationic peptide comprising formula X

wherein R1-R4 are selected from (i) Ac, (ii) H, (iii) H, (iv) H, (i) H,(ii) Ac, (iii) H, (iv) H, (i) H, (ii) H, (iii) Ac, (iv) H, and (i) H,(ii) H, (iii) H, (iv) OH; f) an aromatic-cationic peptide comprisingformula XI

19.-23. (canceled)